Multiple key components of the innate immune system are conserved across eukaryotes. In plants, the innate immune system serves as a barrier to inhibit both pathogen entry and multiplication. Despite the importance of the innate immune system, scientists still have a limited understanding of how plant immune complexes are assembled and regulated in response to pathogen perception. A key regulator of the plant immune system is the Arabidopsis gene RIN4. RIN4 is conserved among all land plants and acts to regulate immune perception of the bacterial pathogen Pseudomonas syringae pv. tomato in Arabidopsis. Preliminary data within this application demonstrate the purification of RIN4 protein complexes in the absence and presence of pathogen stimulus. Fifteen novel proteins were identified by mass spectrometry and multiple proteins were subsequently shown to interact with RIN4 by yeast two-hybrid. Arabidopsis knockout or overexpression lines for three of these RIN4 associated proteins display altered defense responses to P. syringae pv. tomato, suggesting that they are important components of the plant immune response. One RIN4 associated protein is the H+ATPase AHA1. Experiments indicate that RIN4 can directly regulate AHA1 enzymatic activity. RIN4 can work in concert with AHA1 to regulate leaf stomatal opening during the innate immune responses, thus blocking the entry of bacterial pathogens into the leaf interior. The central hypothesis of the proposed research is that RIN4 complex constituents will be key components controlling innate immune signaling. Several proposed experiments seek to understand RIN4 protein complex assembly and RIN4-mediated cellular signaling cascades using the P. syringae-Arabidopsis pathosystem. This pathosystem is an excellent model system to study eukaryotic innate immune signaling because of the extensive genetic resources available, the fast generation time of Arabidopsis, and the similarities between innate immune systems in plants and other eukaryotes. The specific aims of this research application are the following: 1) Elucidate the mechanism RIN4 uses to regulate plasma membrane H+ATPase activity; 2) Investigate the spatial and temporal components of the RIN4 protein network; 3) Functionally characterize Arabidopsis RIN4 associated proteins. PUBLIC HEALTH RELEVANCE: RIN4 is a central player in the regulation and activation of membrane-localized plant innate immune protein complexes. Multiple RIN4 associated proteins are widely conserved among eukaryotes and several have been implicated in immune signaling in mammals. Because there are significant similarities between innate immune systems in plants and other eukaryotes, we anticipate our findings will be broadly relevant to NIH's mission.